Activity of cefepime, carbapenems and new β-lactam/β-lactamase inhibitor combinations on Enterobacter cloacae complex and Klebsiella aerogenes in Spain (SMART 2016–2022)

Abstract Objectives To analyse the susceptibility profile to cefepime, carbapenems and new β-lactam/β-lactamase inhibitor combinations in Enterobacter cloacae complex and Klebsiella aerogenes isolated from intra-abdominal, urinary, respiratory and bloodstream infections in the SMART (Study for Monitoring Antimicrobial Resistance Trends) surveillance study in Spain. Methods The susceptibilities of 759 isolates (473 E. cloacae complex and 286 K. aerogenes) collected in 11 Spanish hospitals from 2016 to 2022 were analysed following the EUCAST 2023 criteria. Molecular characterization looking for β-lactamase genes was performed through PCR and DNA sequencing analysis. Results E. cloacae complex showed resistance to third-generation cephalosporins in 25% of the cases, whereas K. aerogenes was resistant in 35%. Regarding cefepime, resistance in E. cloacae was higher (10%) than in K. aerogenes (2%). Carbapenems showed >85% activity in both microorganisms. Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam had good activity against these microorganisms (>95%). In contrast, the activity of ceftolozane/tazobactam was lower (80%). A high proportion of the isolates resistant to new β-lactam/β-lactamase inhibitor combinations carried a carbapenemase, mainly OXA-48-like and VIM-1. Conclusions Ceftazidime/avibactam, imipenem/relebactam and meropenem/vaborbactam show high activity against both E. cloacae complex and K. aerogenes isolates recovered in the SMART-Spain study. In contrast, differences have been found in the case of cefepime, showing more activity against K. aerogenes than E. cloacae complex. These results are useful for antimicrobial stewardship programmes and for the implementation of local and national guidelines.


Introduction
Antimicrobial resistance is a major problem worldwide.A recent study has estimated that there were 4.95 million deaths associated with bacterial antimicrobial resistance (AMR), in 2019 alone. 1Of these, 1.27 million deaths were attributable directly to AMR.Another study from the ECDC estimated that, in 2015, there were 671 689 infections by MDR bacteria, causing 33 000 deaths. 2 Remarkably, more than 50% were healthcare-related infections.These types of infections have a major clinical impact because they affect patients with severe comorbidities, in which management is difficult, increasing mortality. 3n this sense, surveillance programmes can help to develop antimicrobial stewardship programmes, inform treatment decisions, guide national and local policies and clinical guidelines, and direct efforts to develop new treatment options, improving the clinical outcome of the patients. 4The Study for Monitoring Antimicrobial Resistance Trends (SMART) is one of the largest and longest-standing AMR surveillance programmes.It has been operating since 2002, monitoring trends of antimicrobial susceptibility of aerobic and facultative Gram-negative bacilli from intra-abdominal infections, urinary tract infections, lowerrespiratory tract infections and bloodstream infections. 4,5n recent years, Enterobacter species and Klebsiella aerogenes (formerly Enterobacter aerogenes) have acquired relevance among the microorganisms causing healthcare-related infections due to their ability to develop AMR. 6The presence of an inducible chromosomal AmpC β-lactamase confers extended resistance to third-generation cephalosporins (such as ceftazidime, ceftriaxone or cefotaxime) and piperacillin/tazobactam, when it is overexpressed, limiting the therapeutic options for patients. 7efepime is the antibiotic of choice to treat infections caused by these type of microorganisms because of its low ability to induce AmpC β-lactamases. 8Carbapenems also are considered to treat infections by these microorganisms. 8The IDSA recommends using carbapenems when MICs of cefepime are higher than 2 mg/L due to the high risk of co-producing an ESBL enzyme. 8][11] New β-lactam/β-lactamase inhibitor combinations, such as ceftazidime/avibactam, imipenem/relebactam or meropenem/vaborbactam, also could be alternative options for the treatment of Enterobacter spp.and/or K. aerogenes infections, especially when they are hyperproducing AmpC and exhibit carbapenem resistance that is not due to MBLs. 8 Despite these recommendations, there are few recent studies monitoring the activity of these antibiotics against Enterobacter spp.and K. aerogenes. 12,13In this study, we analysed the susceptibility trend to cefepime, carbapenems and new β-lactam/ β-lactamase inhibitor combinations in Enterobacter spp.and K. aerogenes during a 7 year period (2016-2022), focusing also on the molecular profile associated with resistance to these antibiotics.

Bacterial isolates and antimicrobial susceptibility testing
Clinical isolates of E. cloacae complex and K. aerogenes were recovered at 11 Spanish hospitals from 2016 to 2022.Isolates were subsequently shipped to a central laboratory (IHMA, Schaumburg, IL, USA) for identification by MALDI-TOF and antimicrobial susceptibility testing (AST) by broth microdilution, following the standard ISO recommendations.The following antibiotics were tested: piperacillin/tazobactam, ceftazidime, ceftriaxone, cefepime, ertapenem, imipenem, meropenem, ceftolozane/tazobactam, ceftazidime/avibactam, imipenem/relebactam and meropenem/ vaborbactam.The EUCAST 2023 clinical breakpoints were used to analyse the susceptibility/resistance criteria.To see if there is a trend in the susceptibility profile over the years, we ran a weighted least squares analysis accounting for serial data autocorrelation with Joinpoint Regression v.5.1.0.(Statistical Methodology and Applications Branch, Surveillance Research Program, National Cancer Institute, USA).The time trends for each variable were analysed to find whether the slope significantly increased or decreased over time, or on the contrary, stayed steady over time, which indicates no significant differences among all the annual data in the series.We used a first-order autocorrelation parameter estimated from the data with a permutation test as the model selection method. 14

Molecular characterization
Molecular testing was also centralized at IHMA and included all the isolates resistant to at least one of the following antibiotics: imipenem, imipenem/relebactam or ceftolozane/tazobactam.Molecular testing consisted of screening the following β-lactamase genes through PCR and DNA sequencing: class A ESBLs (TEM, SHV, CTX-M, VEB, PER and GES); class C plasmid AmpC (ACC, ACT, CMY, DHA, FOX, MIR and MOX) and carbapenemases (KPC, GES, NDM, IMP, VIM, GIM, SPM and OXA-48-like), as previously described. 15,16

Susceptibility profile to classic antibiotics
Table 1 shows the susceptibility profile of both microorganisms.Around 75% of the E. cloacae complex and 65% of the K. aerogenes isolates were susceptible to third-generation cephalosporins and piperacillin/tazobactam (Table 1).Regarding cefepime, there were notable differences between the microorganisms, despite it having good activity against both: E. cloacae complex remained less susceptible (90%) than K. aerogenes (98%).Overall, carbapenems (ertapenem, imipenem and meropenem) showed very good activity (>90%) whereas ertapenem activity was lower (86%) for E. cloacae complex (Table 1).

Molecular characterization E. cloacae complex
The molecular resistance profile for the E. cloacae complex is presented in Table 2.In summary, 47 of the 473 isolates (10%) were resistant to cefepime.Of these, 26 (55.3%) harboured a carbapenemase gene, 3 (6.4%)an ESBL gene, and another 3 (6.4%) a combination of an ESBL plus a plasmidic AmpC β-lactamase.In the remaining isolates (n = 15, 31.9%)no β-lactamases were found, except for the chromosomal ampC gene encoding the AmpC β-lactamase, characteristic of these microorganisms.Eighteen isolates showed resistance to imipenem and 23 to meropenem.Among these, one and one isolates, respectively, did not carry any carbapenemase gene (Table 2).

K. aerogenes
In the case of K. aerogenes only 5 of the 286 isolates (1.7%) showed resistance to cefepime.Of these, one isolate carried a plasmidic ampC gene, one isolate an NDM carbapenemase, and three isolates did not carry any acquired β-lactamase (Table 3).Ten isolates were resistant to imipenem and seven resistant to meropenem.

Discussion
In this study, we reported data from the SMART surveillance study in Spain that focused on the activity of cefepime, carbapenems Antimicrobial activity in Enterobacter spp.and K. aerogenes and new β-lactam/β-lactamase inhibitor combinations against Enterobacter spp.and K. aerogenes.Around 25% of the E. cloacae complex and around 35% of the K. aerogenes were resistant to third-generation cephalosporins.These data for E. cloacae complex are similar to those reported previously (24.3%), although they are slightly higher than those reported for K. aerogenes (23.3%). 17These resistance percentages have undergone variations over the years for both microorganisms, but without any clear trend (P > 0.05) (Table 1).Regarding cefepime, differences have been observed among both kinds of microorganisms, with E. cloacae complex being more resistant than K. aerogenes (10% versus 2%).When the molecular characterization was performed, 15 of 47 (32%) cefepime-resistant E. cloacae isolates did not carry any β-lactamase, except for the chromosomal AmpC, suggesting that cefepime resistance also could be mediated by this β-lactamase or by another non-enzymatic mechanism, such as porin loss, as has been previously reported. 9,18,19esistance to carbapenems was variable (1%-14%) in E. cloacae complex and K. aerogenes isolates, with meropenem being the most active carbapenem in both microorganisms (Table 1).Molecular characterization found the presence of at least one  20 However, based on our data we did not observe an increase in the carbapenem resistance percentage.This apparent discrepancy may be due to this increment occurring only in Andalusia, and not in the rest of the country, from where a large proportion of our data was collected.
Finally, resistance to new β-lactam/β-lactamase inhibitor combinations in E. cloacae complex and K. aerogenes was low in general, except for ceftolozane/tazobactam (Table 1).It is known that ceftolozane/tazobactam has lower activity in E. cloacae complex and K. aerogenes when AmpC β-lactamase is hyperproduced. 13his is because tazobactam inhibits AmpC β-lactamases less efficiently. 8In the case of ceftazidime/avibactam, only 11 of 759 isolates (1.4%) (10 E. cloacae complex and 1 K. aerogenes) showed resistance, of which all except 2 (1 E. cloacae and 1 K. aerogenes) carried a VIM-1 carbapenemase (Tables 2 and 3).The remaining two isolates did not carry any acquired β-lactamase, except the chromosomal AmpC.Resistance to ceftazidime/avibactam mediated by AmpC β-lactamases has been reported previously, but only in one study with one isolate from a patient treated with cefepime. 21Further studies are therefore needed to determine whether this β-lactamase is involved in resistance to this antibiotic.For imipenem/relebactam and meropenem/vaborbactam the situation was similar to ceftazidime/avibactam.From 18 (2.3%)isolates resistant to imipenem/relebactam and from 7 isolates resistant to meropenem/vaborbactam, only 1 K. aerogenes and 1 E. cloacae complex isolate, respectively, did not have any carbapenemase gene.Thus, ceftazidime/avibactam, imipenem/ relebactam and meropenem/vaborbactam showed high activity in those isolates that do not carry any carbapenemase.Resistance to ceftazidime/avibactam not due to carbapenemases has already been reported in E. cloacae. 22,23This resistance was mainly associated with one or more amino acid deletions in helix H10 of the AmpC β-lactamase.However, to our knowledge, there are no studies describing the associated resistance mechanisms to imipenem/relebactam and meropenem/vaborbactam.Further studies are therefore needed to clarify this issue.
This study has one main limitation, i.e the molecular characterization was based on resistance to only three antibiotics (imipenem, imipenem/relebactam or ceftolozane/tazobactam), which may underestimate the real number of carbapenemaseproducer strains.
In conclusion, there are differences in the microbiological characteristics between E. cloacae complex and K. aerogenes.In general, E. cloacae complex shows a more resistant profile than K. aerogenes, especially with cefepime.However, ceftazidime/ avibactam, imipenem/relebactam and meropenem/vaborbactam combinations have high activity against both microorganisms.More studies are necessary to analyse the molecular mechanisms that drive resistance to these antibiotics.

Table 2 .
Molecular profiles associated with resistant isolates of Enterobacter cloacae

Table 3 .
Molecular profiles associated with resistant isolates of Klebsiella aerogenes in most of them.A recent study has reported a worrying increase in carbapenemase-producing E. cloacae in the south of Spain from 2014 to 2022. carbapenemase